IGEM:Paris Bettencourt 2012/Notebooks/Semantic group

Notebooks	Design	Roadmap	Meetings and to-dos	Protocols	Bibliography	Previous Biosafety iGEM projects

Semantic containment project

---

The idea

We need to prevent our genetic construction from being used by other organism. Since horizontal gene transfer (HGT) can be perform either by conjugation, by transduction, or by transformation, and none of these systems is only dependent of our organism, we cannot assume the fact that HGT is fully avoidable. Semantic containment [1] means that our bacteria won't be able to "speak" with other organism, since they don't speak the same language. The language being DNA. Here, it won't be all DNA that we are going to change, but just 1 stop codon that we are going to change in a normal aa codon, let say the aa 'X', for OUR GMO bacteria. So, in case of HGT, the gene transferred won't be able to be translate correctly, since it has many stop codon instead of the aa 'X'.

What we can use from others

Church Lab try to engineer a strain in order to remove the rarest stop codon in E coli MG1655, which is TAG (amber stop codon, 314 occurrences), and replace it with the most common stop codon which is TAA [2]. Moreover it already exists a tRNA amber suppressor gene, named supD, that replaces amber stop codon with a serine amino-acids [3]. This system used by Anderson et. al has already been used by different IGEM teams, with either Serine (supD) or Tyrosine (tyrS), including Paris and Pekin University, and it turns out that the presence of the tRNA amber suppressor doesn't disturb the strain.

What we have to do

About amino-acyl synthetase of this tRNA^Ser_TAG

I didn't find any information about this enzyme, therefor I supposed that this enzyme is not specific of the tRNA amber suppressor. Since it's still a supposition, we will still have to look for this information, because, a specific enzyme would really increase the robustness of the system.

Making a Choice between Serine and Tyrosine

Figure 1 : Outline of the algorithm used to discriminate S, Y and other amino-acids. p(i) being the probability of having that amino-acid with the codon i, actually all codon have the same probability to occur, which is 1/9 (9 codons possible with 1 mutation of difference). Subst(AA,AAi) stands for the cost of the substitution of an amino-acids and an other, the first one being the one in input, the other corresponding to the codon i.

As we may not have time to test the efficiency of our system with either Serine (S) or Tyrosine (Y), we have to choose. In order to discriminate which of these two amino-acids, I first check their codon usage in E coli K12 (Codon Usage). It turns out that S has 57,88 codons over 1000 codons when Y has 28,59 codons over 1000 codons. Here it would be more interesting to use the S, as we want our genes to contains more amber codon, so the more S we have, the more amber codon we will have. Second I checked the possibilities of the amber codon to reverse to a S, Y or any similar amino-acids. For that I made a program whose an outline is showed on figure 1. The program calculates a score. The higher is the score, the more likely the mutation will maintain the function. So as we don't want to recover the fonction, we want the lowest score between S and Y. I used 3 types of blosum matrix, blosum62, blosum80, blosum100, all gave the same relation between S and Y, ie. S always has a lower score than Y. The blosum matrix is used for the substitution part in the formula Subst(AA,AAi). Therefor S seems to be less robust to mutation, meanings that if a single substitution occurs in the amber codon, it will be more likely that the function of the amino-acid will be change, resulting in an inefficient protein, a priori.

[math]\displaystyle{ Score_{W} = {1\over9} \sum_{i=1}^9 Subst(AA,AA_{i}) }[/math]

Proof of principle

We should design an experiment that will show that this system is functional. For instance, we could imagine a transformation test, in which we have 2 plasmids. One (say p1) with a reporter gene with all serines replaced with amber codon, and another plasmid (say p2), with the tRNA^Ser_TAG. We will then transform either both plasmid or only the first one (p1), and then plate the 2 transformations to reveal the reporter. We're expecting to see the reporter in the first case since it is expressed, thanks to tRNA amber suppressor. As a reporter, we can use an antibiotic resistance gene. We choose Kanamycin.

Going further

Every single synthetic gene we want to construct have to be coded with amber codon instead of any serine codon (TCN and AGY, N being A,T,C,G and Y being T or C, so 6 codons). Moreover, the aminoacyl-transferase, if it is specific to that tRNA^Ser_TAG, should also have TAG codon instead of normal serine codon. This would enhance massively the robustness of the system, if it's possible (apparently not, cf. above).

Ongoing experiments

We are going to test the tRNA^Ser_TAG by inserting TAG amber codon instead of Serine codon in KmR gene. This test will be qualitative.

Plasmid 1 : pSB3C5::Km_amber

We will order 500 bp oligo twice, in order to have the complete gene (BBa_J31002 - KmR), after one step ligation. We found a unique restriction site in the middle of the gene, so we're gonna be able to ligate after digestion, with the restriction enzyme Alw26I.

Figure 2 : Fragments to be ordered. Black dots above are Amber codon replacing serines. 16 occurrences.

All sequences detailed can be seen here

Figure 3 : Final construction

This will be cloned in EcoRI/PstI into a Low/Medium Copy plasmid, pSB3C5, which is Cm resistant, and provided in the Spring 2012 distribution kit, plate 1, well 3C.

Cloning plan

Add " √ : xx/xx/12 initials" when done.

1. Purchase fragments (F1/F2)
2. Digestion of these fragments with Alw26I
3. Ligation of F1/F2 -> KmR
4. Digestion of KmR with EcoRI and SpeI
5. Digestion of double Terminator with XbaI and PstI
6. Ligation of the 2 last products -> (KmR-dT)
7. Digestion of KmR-dT XbaI and PstI
8. Digestion of P(Kat) with EcoRI and SpeI
9. Digestion of RBS with XbaI and PstI
10. Ligation of the 2 last products -> (P(Kat)-RBS)
11. Digestion of KmR-dT XbaI and PstI
12. Digestion of P(Kat)-RBS with EcoRI and SpeI
13. Ligation of the 2 last products -> Km_final (see figure 3)
14. Digestion of pSB3C5 with EcoRI and PstI -> give vSB3C5 (vector)
15. Ligation of KmR and vSB3C5 -> pSB3C5::KmR
16. Plate on LBA + Cm
17. Re-plate on LBA + Cm + Km

Parts used

Parts number	Name	Distribution/Plate/Well	Backbone
BBa_I14034	P(Kat)	2012/2/13B	pSB2K3
BBa_B0034	RBS	2012/1/2M	pSB1A2
BBa_B0015	double terminator	2012/1/23L	pSB1AK3

NB : For Kanamycin, we use the sequence from the part mentioned, but not the part itself, since we change the serines codon.

Plasmid 2 : pSB1A3::supD_T1

In order to have a functional KmR gene, we need the supD gene on another plasmid, which is in the BioBrick : BBa_K228001. I'll add a strong constitutive promoter, part number BBa_J23119 and a terminator, part number BBa_B1006. The construction before insertion into the plasmid is seen on figure 3.

Figure 4 : Fragment that has to be inserted into pSB1A3

The sequence, and parts number can be seen here

Cloning plan

Add " √ : xx/xx/12 initials" when done.

1. Digestion of supD part with EcoRI and SpeI
2. Digestion of Terminator 1 with XbaI and PstI
3. Ligation of the products of ligation -> give product (1)
4. Digestion of (1) with EcoRI and SpeI
5. Digestion of promoter with Xba1 and Pst1
6. Ligation of the 2 last products -> (2)
7. Digestion of pSB1A3 with EcoRI and PstI -> give vSB1A3 (vector)
8. Ligation of product (2) and vSB1A3 -> give pSB1A3::supD_T1
9. Transformation
10. Plate on LBA + Amp

Parts used

Parts number	Name	Distribution/Plate/Well
BBa_R0011	Inducible promoter, by IPTG	2012/1/6G
BBa_K228001	supD (tRNA amber supressor gene)	2012/4/8K
BBa_B1006	terminator	2012/1/4H

References

1. Marliere, P. The farther, the safer : a manifesto for securely navigating synthetic species away from the old living world. System and Synthetic Biology 3, 77-84 (2009). Paper
2. Isaacs, F.J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science (New York, N.Y.) 333, 348-53 (2011). Paper
3. Anderson, J.C., Voigt, C. a & Arkin, A.P. Environmental signal integration by a modular AND gate. Molecular systems biology 3, 133 (2007).Paper